An ESEEM investigation of Cu(II)-Insulin in polycrystalline samples has revealed that the quadrupole parameters for the remote nitrogen of the two equatorial HIS imidazoles bound to copper are similar to those found in type I copper proteins. When Cd(II) ions are allowed to soak into these crystalline samples a dramatic switch is observed in these parameters, which change to values more similar to those found in type II copper sites. FT-ESEEM simulations based on atomic positions derived from Insulin crystal structures show that this switch is due to a change in the hydrogen bonding environment of the remote nitrogen.